Nucleic acid amplification employing the polymerase chain reaction (PCR) is well known, as are assays that include PCR amplification. See U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,965,188, and, generally, PCR PROTOCOLS, a guide to Methods and Applications, Innis et al. eds., Academic Press (San Diego, Calif. (USA) 1990). Homogeneous PCR assays that do not require washing to remove unbound detector reagents or probes and thus can be performed without opening amplification reaction vessels are also well known. Homogeneous PCR assays include both end-point assays, in which amplified product is detected at the end of the amplification reaction, and real-time assays, in which amplified product is detected during some or all of the thermal cycles as the reaction proceeds. See U.S. Pat. Nos. 5,994,056, 5,487,972, 5,925,517 and 6,150,097.
PCR amplification reactions generally are designed to be symmetric, that is, to make double-stranded amplicons by utilizing a forward primer and a reverse primer that are “matched”; that is, they have melting temperatures that are as close as possible, and they are added to the reaction in equimolar concentrations. A technique that has found limited use for making single-stranded DNA directly in a PCR reaction is “asymmetric PCR.” Gyllensten and Erlich, “Generation of Single-Stranded DNA by the Polymerase Chain Reaction and Its Application to Direct Sequencing of the HLA-DQA Locus,” Proc. Natl. Acad. Sci. (USA) 85: 7652-7656 (1988); and U.S. Pat. No. 5,066,584. Asymmetric PCR differs from symmetric PCR in that one of the primers is added in limiting amount, typically 1-20 percent of the concentration of the other primer.
More recently we have developed a non-symmetric PCR amplification method known as “Linear-After-The-Exponential” PCR or, for short, “LATE-PCR.” See Sanchez et al. (2004) PNAS 101: 1933-1938, Pierce et al. (2005) PNAS 102: 8609-8614, and published international patent application WO 03/054233 (3 Jul. 2003), which is incorporated herein by reference in its entirety. LATE-PCR takes into account the actual melting temperatures of PCR primers at the start of amplification, referred to as Tm[0]. Tm[0] can be determined empirically, as is necessary when non-natural nucleotides are used, or calculated according to the “nearest neighbor” method (Santa Lucia, J. (1998) PNAS (USA) 95: 1460-1465; and Allawi, H. T. and Santa Lucia, J. (1997) Biochem. 36: 10581-10594) using a salt concentration adjustment. In our work we have utilized 0.07M monovalent salt concentration.
An undesirable feature of PCR amplifications, reduced in the case of LATE-PCR, is scatter among replicates. Following the exponential phase of the amplification, replicate amplifications followed in real time diverge and plateau at different levels. Scatter indicates that replicates do not have the same reaction kinetics and reduces accuracy. This is a problem for PCR assays generally, but particularly for end-point assays and assays that depend upon the slope of signal during the linear phase.
Another significant problem with PCR amplifications is mispriming, which we believe is manifest in at least three types: Type 1, mispriming that occurs during preparation of reaction mixtures prior to the start of amplification; Type 2, mispriming that occurs during amplification if cycle temperatures include any temperature significantly below the melting temperature of a primer; and Type 3, mispriming that occurs in the late stages of a PCR amplification that is continued after a high concentration of amplicon has been made. Several approaches have been used to address the first type of mispriming. One approach is to modify the polymerase chemically so that it is inactive until heated to a high temperature such as 95° C. See U.S. Pat. Nos. 5,677,152 and 5,773,258. Another approach is to bind an antibody to the polymerase to inhibit the polymerase until the reaction is heated to a high temperature such as 95° C. to irreversibly denature the antibody. See U.S. Pat. No. 5,338,671. Yet another approach is to include an aptamer in the reaction mixture. See Doug and Jayasena (1996), J. Mol. Biol. 264: 268-278 and U.S. Pat. No. 6,020,130. An aptamer is a single-stranded oligonucleotide approximately 30 nucleotides in length that binds to a polymerase and inhibits its ability to extend a recessed 3′ end at low temperatures. Aptamers are not irreversibly denatured at 95° C., a typical highest temperature for a PCR cycle. Kainz et al. (2000) Biotechniques 28: 278-282 reported that the addition to PCR reaction mixtures of double-stranded DNA fragments having lengths of 16-21 nucleotides in certain amounts inhibit polymerases at temperatures below typical PCR extension temperatures and suppress synthesis of non-specific products. DNA fragments are not irreversibly denatured during PCR cycling. Eppendorf-5 Prime, Inc. markets a proprietary ligand that is said to bind to Taq polymerase in a temperature-dependent manner and to inhibit its binding to double-stranded DNA at temperatures below about 50° C. Despite these many attempts, mispriming remains a problem with PCR amplifications.
Another manifestation of mispriming during PCR amplification is known as primer-dimer formation and amplification. According to this phenomenon one primer hybridizes to the other primer or to itself and then undergoes extension of the 3′ end to generate a small double-stranded amplicon, which can then amplify further or can multimerize and amplify further. Primer-dimer formation can occur in the absence of target.
Quantitative analysis of PCR amplifications has been enabled by real-time detection methods, as the PCR cycle at which fluorescent signal becomes visible above the threshold cycle or CT of reactions is indicative of starting target concentrations. End-point analyses are semi-quantitative at best, due in part to scatter among replicates as the reaction exits exponential amplification. Electrophoretic analysis of double-stranded amplicons is semi-quantitative, and may utilize fluorescently labeled primers. End-point analysis utilizing fluorescently labeled probes, either allele-discriminating probes or mismatch-tolerant probes, are also semi-quantitative at best. By reducing scatter and producing single-stranded product, LATE-PCR offers significant improvement in end-point analysis, but scatter among replicates is often not completely eliminated, leaving quantitative multiplex detection less accurate than desired.
An aspect of this invention is a class of reagent additives to improve product specificity and to eliminate the effects of mispriming in PCR amplification reactions. These additives out-perform existing “hot-start” methodologies in all types of PCR and can be used to prevent the accumulation of undesired products, including primer-dimers and misprimed amplicons, both at early stages of the reaction and during LATE-PCR reactions having many cycles (typically 60 cycles and more).
Another aspect of this invention is PCR amplification and assay methods, both symmetric PCR or non-symmetric PCR, including but not limited to LATE-PCR, and kits, partial kits, and oligonucleotide sets that include such reagent additives.